We will investigate the number, nature, organization and function of Agrobacterium tumefaciens genes involved in crown gall tumorigenesis. We will do this by determining the defects in mutants specifically blocked in their ability to induce tumors. Such mutants will be isolated as a consequence of transposon insertion and deletion events. Mutants will be characterized with respect to physiological, biochemical and genetic parameters. The site of the mutation will be ascribed to either the chromosome or the Ti plasmid by genetic techniques. Mutations in the Ti plasmids will be physically mapped by restriction enzyme analysis and functionally mapped by genetic complementation analysis. The latter will be accomplished by using fragments of wild-type or mutant Ti plasmids cloned in A. tumefaciens vector plasmid. We are developing this vector plasmid from pAgK84, the plasmid responsible for agrocin 84 biosynthesis. Chromosomal mutations will be mapped by conjugal transfer, using sex factors we are constructing from the IncP1 R plasmid R68-45. We will use the ability of some of our mutants to phenotypically complement each other for tumor formation to determine events involved in bacterial infection and transfer of the Ti plasmid to host plant cells. We will determine, using genetically marked strains, which mutant of a given pair actually transfers its Ti plasmid. We will analyze the state of tDNA in slow-growing tumors induced by certain of our mutants unable to strongly site-bind. This should provide information concerning tDNA transfer, processing and functions. Finally, we will develop a physical and genetic map of pAgK84. We will also construct derivatives of this plasmid suitable for use as cloning vectors in Agrobacterium.